A technique of bone marrow collection from vertebral bodies of cynomolgus macaques for transplant studies

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Abstract

Strategies to induce donor-specific allograft tolerance are best tested in preclinical models developed in nonhuman primates (NHPs). Most protocols prepare the recipient by infusing hematopoietic cells from the donor. We report here a procedure to isolate and characterize large numbers of bone marrow cells (BMCs) from cynomolgus monkeys (cynos) that can then successfully be transplanted into conditioned recipients.Vertebral columns of five cynos were excised en bloc and separated into individual vertebrae. The cancelous bone was extracted with a core puncher, fractionated, filtered, centrifuged, and resuspended in transplantation media before being analyzed by flow cytometry. In two instances, the collected BMCs were reinfused into allogeneic recipients preconditioned with a nonmyeloablative regimen. Chimerism was monitored using short-tandem repeat analysis.The mean total BMCs yield was 25.5 x 10(9) (range of 4.00 x 10(9) to 59 x 10(9)) with mean cell viability of 93.4% (range: 90-96%). CD34+ cells and CD3+ cells averaged 0.34 and 3.91% of total BMCs, respectively. This resulted in absolute cell number yields of 1.02 x 10(8) and 1.15 x 10(9) for CD34+ and CD3+ cells, respectively. Graft-versus-host disease was absent in both bone marrow infused animals, and a maximum level of chimerism of 18% was detected at 3 weeks after BMCs infusion.We present here the first detailed report of a procedure to retrieve and characterize large numbers of BMCs from vertebral bodies of cynos and demonstrate that cells collected with this technique have the capability of engrafting in allogenic recipients.

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